http://dx.doi.org/10.1590/1519-6984.

01015  

Morphometric differences and fluctuating asymmetry in

Melipona subnitida Ducke 1910 (Hymenoptera: Apidae)
in different types of housing
C. B. S. Limaa, L. A. Nunesb*, C. A. L. Carvalhoa, M. F. Ribeiroc,
B. A. Souzad and C. S. B. Silvaa
a
Programa de Pós-graduação em Ciências Agrárias – PRPPGCA, Universidade Federal do Recôncavo da Bahia – UFRB,
Rua Rui Barbosa, 710, Centro, CEP 44380-000, Cruz das Almas, BA, Brazil
b
Programa de Pós-graduação em Genética Biodiversidade e Conservação – PPGGBC, Universidade Estadual do Sudoeste
da Bahia – UESB, Rua José Moreira Sobrinho, s/n, Jequiezinho, CEP 45206-190, Jequié, BA, Brazil
c
Setor de Entomologia, Empresa Brasileira de Pesquisa Agropecuária – Embrapa, Rodovia BR-428, Km 152,
Zona Rural, CP 23, CEP 56302-970, Petrolina, PE, Brazil
Setor de Apicultura, Empresa Brasileira de Pesquisa Agropecuária – Embrapa, Av. Duque de Caxias, 5650,
d

Bairro Buenos Aires, CP 001, CEP 64006-220, Teresina, PI, Brazil

*e-mail: lorenunes2@gmail.com

Received: January 13, 2015 – Accepted: August 26, 2015 – Distributed: November 30, 2016
(With 3 figures)

Abstract
A geometric morphometrics approach was applied to evaluate differences in forewing patterns of the Jandaira bee
(Melipona subnitida Ducke). For this, we studied the presence of fluctuating asymmetry (FA) in forewing shape and
size of colonies kept in either rational hive boxes or natural tree trunks. We detected significant FA for wing size as
well as wing shape independent of the type of housing (rational box or tree trunks), indicating the overall presence
of stress during the development of the studied specimens. FA was also significant (p < 0.01) between rational boxes,
possibly related to the use of various models of rational boxes used for keeping stingless bees. In addition, a Principal
Component Analysis indicated morphometric variation between bee colonies kept in either rational hive boxes or in
tree trunks, that may be related to the different origins of the bees: tree trunk colonies were relocated natural colonies
while rational box colonies originated from multiplying other colonies. We conclude that adequate measures should
be taken to reduce the amount of stress during bee handling by using standard models of rational boxes that cause the
least disruption.
Keywords: beekeeping, geometric morphometrics, shape, wing, bee handling.

Diferenças morfométricas e assimetria flutuante em Melipona subnitida

Ducke 1910 (Hymenoptera: Apidae) em diferentes tipos de habitação

Resumo
A abordagem da morfometria geométrica foi aplicada para avaliar as diferenças nos padrões das asas anteriores da
abelha Jandaíra (Melipona subnitida Ducke). Para isso, estudou-se a presença de assimetria flutuante (AF) na forma
das asas anteriores e tamanho das colônias mantidas tanto em caixas de colméia racional ou troncos de árvores naturais.
Foi detectado AF significativa para o tamanho da asa, bem como a forma da asa independente do tipo de habitação
(caixa racional ou cortiço), indicando a presença global de estresse durante o desenvolvimento dos espécimes estudados.
AF também foi significativa (p < 0,01) entre as caixas racionais, possivelmente relacionados com a utilização de vários
modelos de caixas racionais utilizados para a conservação de abelhas sem ferrão. Além disso, a Análise de Componentes
Principais indicou variações morfométricas entre as colônias de abelhas mantidos em caixas racionais ou em cortiços,
que podem estar relacionados com as diferentes origens das abelhas: os cortiços foram colônias naturais realocados,
enquanto as colônias das caixas racionais foram originadas da multiplicação outras colônias. Conclui-se que devem ser
tomadas medidas adequadas para reduzir a quantidade de estresse durante o manuseio abelha usando modelos padrão
de caixas racionais que causam a menor perturbação.
Palavras-chave: meliponicultor, morfometria geométrica, forma, asa, manejo.

Braz. J. Biol.       1
 Lima, C.B.S. et al.
1. Introduction
Melipona subnitida occurs naturally in the savannah region
of north-eastern Brazil. Commonly used for beekeeping,
rational hive boxes are advantageous for bee handling
and high yields of honey and other products (Bruening,
2001; Cortopassi-Laurino and Imperatriz-Fonseca, 2001;
Lopes et al., 2007; Camargo and Pedro, 2013).
Meliponiculture, the cultivation of native stingless bees
in rational hives has been practiced as a leisure activity
as well as to exploit hive products as an income source,
among other motives. Moreover, it has contributed to the
preservation of species (Villas-Boas, 2012).
Traditionally, bees are kept and reared in tree hollows
where the colonies naturally build their nests (Nogueira‑Neto,
1953; Buchmann, 2006). However, the capture of bee
colonies prior to collecting hive products is considered
aggressive, and causes stress on the bee colony that results
from various disturbances in the nest, including damage
to structures, displacement of food pots, loss of larvae
and sometimes the death of the queen (Alves et al., 2005).
Alternatively, bees can be kept in wooden rational hive
boxes, size-specific for each species (Nogueira-Neto, 1953;
Buchmann, 2006). Bee development in rational boxes
may be influenced by using material that is inappropriate
for bee keeping. However, in order to provide an efficient
management, beekeepers attempt to adjust boxes for the
characteristics of each bee species, taking into account
available information about nest architecture and bee
biology (Souza et al., 2009).
There are several models of rational boxes for keeping
stingless bees. The most suitable one for rearing and
reproduction of bees of the Melipona genus is a vertical
box, designed by the National Research Institute of
Amazonia-INPA. This model allows for less interference
by the meliponicultor, permits the colony to recover,
and facilitates management by the “Method of Minimal
Disruption” (Oliveira and Kerr, 2000).
Despite proper handling, bees experience stress
during and after the transfer of colonies to rational boxes
especially when opening the tree trunk for nest removal,
an aggressive procedure likely to cause breakage of food
pots and crushing of young worker bees (Nogueira-Neto,
1997). According to this author, stress to the bees is also
caused by the process of honey collection itself: opening
the box creates an excess of moisture that interferes
with offspring development, produces an imbalance in
thermoregulation of the colony and promotes a suitable
environment for fungi, among other damages.
The induced stress can impact the ontogenetic
development of individuals resulting in asymmetries.
Fluctuating asymmetry (FA) is characterized by normally
distributed deviations from the bilateral body symmetry
of individuals. The presence of differences between the
right and left body side is considered an indicator of stress
(Clarke, 1998) and is a widely used parameter to evaluate
instability and plasticity caused by stressful conditions
during the development of organisms (Graham et al., 2010).
Rather than being genetically determined, this parameter
is mostly influenced by environmental characteristics that
2 Braz. J. Biol.
affect the ontogeny of individuals (Leamy and Klingenberg,
2005). In addition, FA reflects the degree of population
adaptation, making it an important tool for studying the
biology of populations (Graham et al., 2010).
Genetic or environmental disturbances that organisms
may encounter during their embryonic development must
be overcome to allow the expression of the phenotype
pre-determined for the species, and can be evaluated by
FA. Developmental homeostasis results from the joint
action of genes, producing a developmental pattern
(Del Lama et al., 2002). Because of the rapid and intense
environmental change caused by anthropogenic activities,
such as deforestation and pollution, there is growing concern
about the ecological and evolutionary consequences of
human activities on natural populations (Polak et al., 2002).
Given the need for information about the effect of
stress on stingless bee colonies, the present study aims to
examine fluctuating asymmetry based on the variation in
wing shape and wing size in individuals of M. subnitida
kept in either rational boxes or trunks.
2. Material and Methods
2.1. Study area
Sampling was conducted in meliponaries in the states of
Alagoas, Bahia and Pernambuco. A total of 634 specimens
was collected from 59 nests (41 from rational boxes and
18 from tree trunks). The specimens were placed in falcon
tubes containing 70% alcohol and stored at –20° C.
2.2. Image acquisition
For imaging, the anterior right and left wings were
removed with tweezers and subsequently placed between
microscope slides. Photographs were taken with a digital
camera mounted on a stereomicroscope for the analysis
of venation patterns.
The captured images were transformed to tpsUtil software
version 1.40 (Rohlf, 2008a). Ten anatomical landmarks
(Figure 1) were inserted at the vein junctions of each wing
using tpsDig version 2.17 (Rohlf, 2008b). The images of
left wings were mirrored to achieve identical position
for left and right wings, facilitating the measurement of
anatomical landmarks. For each wing, measurements were
duplicated in order to account for measurement errors
(Palmer, 1994). The data points obtained were used as
variables for statistical analysis.
2.3. Data analysis
To evaluate variations in centroid size and shape of
wings, a Procrustes ANOVA (Klingenberg and McIntyre,
1998; Palmer and Strobeck, 2003) was conducted with
centroid size and shape used as independent variables,
body side as fixed effect and individual as random effect
(Klingenberg and McIntyre, 1998). Thus, the effect of
individual represents individual variation in shape, and the
effect of body side represents wing asymmetry. Analyses
were performed using the software MorphoJ.
The value for the effect of body side and of individuals
was obtained by the denominator of the interaction of
      
 Morphometric differences and asymmetry in Melipona subnitida

body side X individuals obtained by the denominator of
the measurement error (Klingenberg and McIntyre, 1998).
Based on the resulting data, a second ANOVA was
performed in R using the Procrustes coordinates of wings
to evaluate differences and levels of asymmetry between
colonies reared in rational boxes and in tree trunks.
Variation analysis of morphometric divergence for
the evaluation of differences between colonies kept in
rational boxes and in tree trunks was based on matrices
obtained from the anatomical landmark coordinates of
the right forewing, which were processed by Procrustes
overlay plots, and subsequently analyzed using MANOVA.
A covariance matrix was generated for the Principal
Component Analysis (PCA). Analyses were performed
using the software MorphoJ (Klingenberg, 2011).
3. Results
The significant interaction of individual X side (indicating
individual variation in size and shape), confirmed the
presence of significant wing asymmetry within populations
from both types of housing (Tables 1 and 2, p < 0.01).

Figure 1. Right forewing of Melipona subnitida with 10 anatomical landmarks scored in the vein junctions and used for
morphometric analysis.

Table 1. ANOVA Procrustes analysis of wing size and shape of rational hive box colonies with a significant interaction
between the effects of individual and body side.
Effect SS MS df F P (param.)
Individual 156.70 0.36 427 560.72 <.0001
Centroid Size Side 0.00 0.00 1 0.00 0.99
Ind*Side 0.27 0.00 427 9.68 <.0001
Error 0.05 0.00 856
Effect SS MS df F P (param.)
Individual 0.79 0.00 6832 5.52 <.0001
Shape Side 0.00 0.00 16 26.69 <.0001
Ind*Side 0.14 0.00 6832 2.19 <.0001
Error 0.13 0.00 13696

Table 2. ANOVA Procrustes analysis of wing size and shape of tree trunk colonies with a significant interaction between the
effects of individual and body side.
Effect SS MS df F P (param.)
Individual 4.50 0.02 198 32.89 <.0001
Centroid Size Side 0.00 0.00 1 0.98 0.3229
Ind*Side 0.13 0.00 198 12.01 <.0001
Error 0.02 0.00 398 0.03 1.00
Residual 0.09 0.00 44
Effect SS MS df F P (param.)
Individual 0.35 0.00 3168 5.38 <.0001
Shape Side 0.00 0.00 16 6.15 <.0001
Ind*Side 0.06 0.00 3168 2.30 <.0001
Error 0.05 0.00 6368 0.30 1.00
Residual 0.02 0.00 704

Braz. J. Biol.       3
 Lima, C.B.S. et al.

For wing shape, we detected the presence of directional The means for wing shape of Melipona subnitida
asymmetry (p < 0.001), but not for wing size (p > 0.05), colonies kept in different types of housing were plotted
regardless of the type of housing (Table 1 and 2). in two-dimensional space formed by the scores of the first
Although fluctuating asymmetry in wing form and size two principal components explaining 23.84% and 17.92%
was detected in colonies from rational hive boxes and as of the variance, respectively (Figure 3).
well as from tree trunks, there was no significant difference
(p > 0.05) between the two (Figure 2 and Table 3).
Table 3. ANOVA results for fluctuating asymmetry
The first four components of a Principal Component
comparisons between two types of bee housing (rational
Analysis (used to evaluate morphometric divergence between hive boxes and natural tree trunks).
colonies from the two housing types) explained 65.64% of
Df Sum Sq Mean Sq F value Pr(>F)
the total variation between colonies (PC1 (23.84%), PC2
(17.92%) PC3 (1.14%) and PC4 (9.85%)). Morphometric Habitat 1 0.00 2.3263e-05 1.39 0.23
variation was independent of the proximity of colonies, Colonies 60 0.00 3.5782e-05 2.15 0.00
bearing in mind that colonies in rational boxes and tree
Residuals 565 0.00 1.6620e-05
trunks were kept in the same meliponary.

Figure 2. Box plot showing variation of fluctuating asymmetry between nesting environments.

Figure 3. Principal component analysis of wing shape (means) of Melipona subnitida colonies kept in different types of
housing, with the first and second principal axes shown (PC1, PC2).

4 Braz. J. Biol.      
 Morphometric differences and asymmetry in Melipona subnitida
4. Discussion
The significant interaction of individual X side suggest
that stress levels caused by environmental factors such as
minimal variations in temperature, sunlight, and scarcity
of food resources (Ricklefs, 2009) may directly influence
wing size and wing shape of the bees, regardless of their
housing conditions. Wing shape is more likely to be
influenced by the environment than wing size, where only
one of them may be sufficient to indicate the presence of
stress during individual development (Nunes et al., 2013).
Smith et al. (1997) suggest that directional asymmetry
in bees may be related to the position of larvae, pupae and
pre-pupae in the brood cell. It is known that stingless bees
kept in either tree trunks or rational hive boxes suffer a
certain amount of stress related to the type of housing.
Tree trunk colonies may be mainly subjected to environmental
stress such as temperature variation and food shortages,
while rational hive box colonies are usually subjected to
stress caused by human activities such as the frequency
of opening the boxes for artificial food supply in times of
food shortage, or the harvest of honey (Kerr et al., 1996).
Several factors such as heat, lack of food, exposure to sun
and rain may exert stress on individuals, thus explaining
the variation observed in our results.
Stress caused to bees in rational boxes or trunks
may vary during the removal of colonies from the field,
transport to the meliponary, and subsequently during the
honey gathering process by destruction of food pots and
sometimes the death of the queen, the transfer of colonies
into boxes, the opening of boxes for feeding, during colony
division and related to other characteristics of the boxes
and the meliponaries where they are installed.
Although fluctuating asymmetry in wing form and
size was detected in colonies from rational hive boxes
and as well as from tree trunks, this variation may be
related to the use of various models of rational boxes for
keeping stingless bees. The box most suitable for rearing
and multiplication of bees of the genus Melipona is the
INPA model, considered to involve less “meliponicultor”
interference during colony recovery and to allow management
by the “Method of Minimum Disturbance” (Oliveira and
Kerr, 2000). Despite this, most of the rational boxes from
which samples were collected were of a different model.
The morphometric divergence between colonies kept
in boxes or rational in tree trunks may be explained by the
difference in management between the different housing
types and management methods. The colonies found in
tree trunks are natural colonies, collected in the field and
relocated to meliponary generally do not suffer from stress
caused by human activities, however, while the colonies
kept in rational boxes are obtained by multiplication and
division of transferred colonies. The practice of nest
multiplication increases the number of colonies within a
short period of time, and is widely used due to an interest in
the marketing of bee products (Aidar and Campos, 1998).
Braz. J. Biol.       5
Fluctuating asymmetry detected in bee wings can be
attributed to the presence of stress affecting the colonies,
related to handling procedures for each housing type.
Acknowledgements
The authors thank Empresa Brasileira de Pesquisa Agropecuária
(EMBRAPA) (Cod. SEG 02.11.01.029.00.00) for financial
support, Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) for the scholarships (C. B. da
S. Lima) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) (Proc. 305228/2013-7)
and the beekeepers who provided samples of bees.
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